Novel human kunitz-type inhibitors and methods relating thereto

ABSTRACT

The present invention provides isolated DNA molecules comprising a DNA segment encoding novel human Kunitz-type inhibitors. Also provided are DNA constructs comprising a first DNA segment encoding a novel human Kunitz-type inhibitor wherein said first DNA segment is operably linked to additional DNA segments required for the expression for the first DNA segment, as well as host cells containing such DNA constructs and methods for producing proteins from the host cells.

The present invention is divisional of U.S. patent application Ser. No.10/680,684, filed Oct. 7, 2003, which is a divisional of U.S. patentapplication Ser. No. 09/904,621, filed Jul. 13, 2001, now U.S. Pat. No.6,656,746, which is a continuation of U.S. patent application Ser. No.09/265,627, filed Mar. 9, 1999, now abandoned, which is a divisional ofU.S. Pat. application Ser. No. 08/457,887, filed Jun. 1, 1995, now U.S.Pat. No. 5,914,315, which is a divisional of U.S. patent applicationSer. No. 08/147,710, filed Nov. 5, 1993, now U.S. Pat. No. 5,455,338,all of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

Blood coagulation is a process consisting of a complex interaction ofvarious blood components, or factors, which eventually gives rise to afibrin clot. Generally, the blood components that participate in whathas been referred to as the coagulation “cascade” are proenzymes orzymogens, enzymatically inactive proteins that are converted toproteolytic enzymes by the action of an activator, itself an activatedclotting factor. Coagulation factors that have undergone such aconversion are generally referred to as “active factors,” and aredesignated by the addition of a lower case postscript “a” (e.g., factorVIIa).

Two systems promote blood clotting and thereby participate in normalhemostasis. These systems have been referred to as the “intrinsic” andthe “extrinsic” coagulation pathways. It is now believed that theintrinsic pathway plays a role in the growth and maintenance of fibrinformation and that the “extrinsic” pathway is an overlapping mechanismthat is critical for the initiation of fibrin formation. The pathwaysconverge at the activation of factor X to Xa and proceed through a“common” pathway to fibrin formation. After vascular injury, tissuefactor initiates the “extrinsic” coagulation pathway by complexing withfactor VII in a calcium-dependent manner to facilitate the conversion offactor VII to VIIa. The factor VIIa-tissue factor complex can directlyactivate factor X to Xa. The intrinsic pathway may be activated by thegeneration of thrombin or factor XIIa which cleaves factor XI togenerate factor XIa, the required enzyme for the initiation of the“intrinsic” coagulation cascade.

Fibrin formation via the “extrinsic” pathway is controlled by thepresence of tissue factor pathway inhibitor protein (TFPI) whichregulates the pathway in a factor Xa-dependent manner. TFPI, amultivalent Kunitz-type inhibitor, is believed to regulate the extrinsicpathway by forming a quaternary complex with factor Xa, tissue factorand factor VIIa, thus inhibiting the formation of free factor Xa andfactor VIIa (Broze et al., Biochemistry 29: 7539-7546, 1990; which isincorporated by reference herein in its entirety).

In some instances, for example, kidney dialysis, deep vein thrombosis,and disseminated intravascular coagulation (DIC), it is necessary toblock the coagulation cascade through the use of anticoagulants, such asheparin, coumarin, derivatives of coumarin, indandione derivatives, orother agents. A heparin treatment or an extracorporeal treatment withcitrate ion (U.S. Pat. No. 4,500,309) may, for example, be used indialysis to prevent coagulation in the course of treatment. Heparin isalso used in preventing deep vein thrombosis in patients undergoingsurgery. Treatment with low doses of heparin may, however, cause heavybleeding. Furthermore, because heparin has a half-life of approximately80 minutes, it is rapidly cleared from the blood. Because heparin actsas a cofactor for antithrombin III (AT III), and antithrombin III israpidly depleted in DIC treatment, it is often difficult to maintain theproper heparin dosage, necessitating continuous monitoring of AT III andheparin levels. Heparin is also ineffective if AT III depletion isextreme. Further, prolonged use of heparin may increase plateletaggregation, reduce platelet count, and has been implicated in thedevelopment of osteoporosis. Indandione derivatives may also have toxicside effects.

In addition to the anticoagulants briefly described above, there are avariety of compositions disclosed within the art that are alleged tohave anticoagulant activity. One such composition is disclosed byReutelingsperger et al. (Eur. J. Biochem. 151: 625-629, 1985) whoisolated a 32,000 dalton polypeptide from human umbilical cord arteries.Another composition is disclosed by Warn-Cramer et al. (CirculationSuppl, part 2, 74: 2-408ii, Abstract #1630, 1986). They detected afactor VIIa inhibitor of an apparent molecular weight of 34,500 inplasma.

Protein inhibitors are classified into a series of families based onextensive sequence homologies among the family members and theconservation of intrachain disulfide bridges (for review, see Laskowskiand Kato, Ann. Rev. Biochem. 49: 593-626, 1980). Serine proteaseinhibitors of the Kunitz family are characterized by their homology withaprotinin (bovine pancreatic trypsin inhibitor). Aprotinin is known toinhibit various serine proteases including trypsin, chymotrypsin,plasmin and kallikrein. Kunitz-type inhibitor domains have been reportedin larger proteins such as the inter-α-trypsin inhibitors (Hochstrasseret al., Hoppe-Seylers Z. Physiol. Chem. 357: 1659-1661, 1969 andTschesche et al., Eur. J. Biochem. 16: 187-198, 1970), the β-amyloidprotein precursor and the α₃-collagen type VI (Chu et al., EMBO J. 9:385-393, 1990). TFPI (also known as extrinsic pathway inhibitor (EPI) orlipoprotein-associated coagulation inhibitor (LACI)) is a plasmaprotease inhibitor that consists of three tandem Kunitz-type inhibitorsflanked by a negatively charged amino terminus and a positively chargedcarboxyl terminus. The first and second Kunitz-type domains have beenshown to inhibit factor VIIa and factor Xa activity, respectively.

There is still a need in the art for improved compositions havinganticoagulant activity that do not produce the undesirable side effectsassociated with traditional anticoagulant compositions. The presentinvention fulfills this need, and further provides other relatedadvantages.

It is therefore an object of the present invention to provide novelhuman protease inhibitors of the Kunitz family of inhibitors withsimilar inhibitor profiles for use as anticoagulants and in thetreatment of deep vein thrombosis and DIC.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides DNA molecules whichcomprise a DNA segment encoding a Kunitz-type inhibitor, wherein the DNAsegment comprises the sequence of nucleotides of SEQ ID NO:14 from 1 to165, wherein each nucleotide triplet 1 to 3, 4 to 6, 160 to 162 and 163to 165 individually encodes any amino acid except cysteine. Within oneaspect of the invention, the Kunitz-type inhibitor comprises thesequence of nucleotides of SEQ ID NO:1 from nucleotide 138 to nucleotide305. Within another aspect of the invention, the Kunitz-type inhibitorcomprises the sequence of nucleotides of SEQ ID NO:1 from nucleotide 39to nucleotide 743. Within another aspect, the Kunitz-type inhibitorcomprises the sequence of nucleotides of SEQ ID NO:1 from nucleotide 138to nucleotide 493. Within yet another aspect of the invention, theKunitz-type inhibitor comprises the sequence of nucleotides of SEQ IDNO:1 from nucleotide 138 to nucleotide 671.

Within one aspect of the invention, the DNA segment encodes aKunitz-type inhibitor comprising the amino acid sequence of SEQ ID NO:15wherein each Xaa is individually any amino acid except cysteine. Withinone aspect of the invention, the DNA segment encodes a Kunitz-typeinhibitor comprising the amino acid sequence of SEQ ID NO:2 fromglutamic acid, amino acid number 34 to isoleucine, amino acid number 89.Within another aspect of the invention, the DNA segment encodes aKunitz-type inhibitor comprising the amino acid sequence of SEQ ID NO:2from Met, amino acid 1 to Phe, amino acid number 235. Within anotheraspect of the invention, the DNA segment encodes a Kunitz-type inhibitorcomprising the amino acid sequence of SEQ ID NO:2 from glutamic acid,amino acid number 34 to lysine, amino acids 152. Within yet anotheraspect of the invention, the DNA segment encodes a Kunitz-type inhibitorcomprising the amino acid sequence of SEQ ID NO:2 from glutamic acid,amino acid number 34 to alanine, amino acid number 211.

The present invention also provides DNA constructs comprising a firstDNA segment encoding a human Kunitz-type inhibitor operably linked toadditional DNA segments necessary for the expression of the first DNAsegment, host cells containing such DNA constructs, as well as methodsfor producing a human Kunitz-type inhibitor comprising the step ofculturing a host cell and isolating said Kunitz-type inhibitor.

Within another aspect of the invention, isolated Kunitz-type inhibitorsare provided. Within another embodiment, an isolated human Kunitz-typeinhibitor comprises the amino acid sequence of SEQ ID NO:15 wherein eachXaa is individually any amino acid except cysteine. Within one aspect ofthe invention, the Kunitz-type inhibitor comprises the amino acidsequence of SEQ ID NO:2 from Met, amino acid 1 to Phe, amino acid number235; the amino acid sequence of SEQ ID NO:2 from glutamic acid, aminoacid number 34, to isoleucine, amino acid number 89; the amino acidsequence of SEQ ID NO:2 from glutamic acid, amino acid number 34 tolysine, amino acid number 152 or the amino acid sequence of SEQ ID NO:2from glutamic acid, amino acid number 34 to alanine, amino acid number211. Within another aspect of the invention, the Kunitz-type inhibitorfurther comprises the amino acid sequence of SEQ ID NO:12 or SEQ IDNO:13 at its amino-terminus.

Within another aspect of the invention, isolated antibodies are providedwhich specifically bind to a human Kunitz-type inhibitor. Within oneembodiment, the antibody is a monoclonal antibody.

Within yet another aspect of the invention, a pharmaceutical compositionis provided which comprises the amino acid sequence of SEQ ID NO:15wherein each Xaa is individually any amino acid except cysteine. Withinone aspect of the invention, the pharmaceutical composition comprises ahuman Kunitz-type inhibitor comprising the amino acid sequence of SEQ IDNO:2 from Met, amino acid 1 to Phe, amino acid number 235; the aminoacid sequence of SEQ ID NO:2 from glutamic acid, amino acid number 34,to isoleucine, amino acid number 89; the amino acid sequence of SEQ IDNO:2 from glutamic acid, amino acid number 34 to lysine, amino acidnumber 152 or the amino acid sequence of SEQ ID NO:2 from glutamic acid,amino acid number 34 to alanine, amino acid number 211.

Within yet another aspect of the invention, a method for inhibitingblood coagulation in a mammal is disclosed comprising administering ahuman Kunitz inhibitor, comprising the amino acid sequence of SEQ IDNO:15 wherein each Xaa is individually any amino acid except cysteine,in an amount sufficient to inhibit blood coagulation. Within anotheraspect of the invention, a method for inhibiting blood coagulation in amammal is disclosed in which a Kunitz-type inhibitor comprises the aminoacid sequence of SEQ ID NO:2 from methionine, amino acid 1 tophenylalanine, amino acid number 235; the amino acid sequence of SEQ IDNO:2 from glutamic acid, amino acid number 34 to isoleucine, amino acidnumber 89; the amino acid sequence of SEQ ID NO:2 from glutamic acid,amino acid number 34 to lysine, amino acids 152 or the amino acidsequence of SEQ ID NO:2 from glutamic acid, amino acid number 34 toalanine, amino acid number 211 is administered in an amount sufficientto inhibit blood coagulation. In yet another aspect of the invention, amethod for inhibiting blood coagulation in a mammal is provided in whicha Kunitz-type inhibitor further comprises the amino acid sequence of SEQID NO:12 or SEQ ID NO:13 at its amino-terminus, is administered in anamount sufficient to inhibit blood coagulation.

Within another aspect of the invention, probes of at least 12nucleotides are provided, wherein the probes are capable of hybridizingwith nucleic acids encoding a Kunitz-type inhibitor domain comprisingthe nucleotide sequence of SEQ ID NO:1, nucleotide variants of SEQ IDNO:1, or DNA segments encoding DNA sequences complementary to SEQ IDNO:1 or its variants.

These and other aspects will become evident upon reference to thefollowing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel human Kunitz-type inhibitors. Oneadvantage of the inhibitors of the present invention is that theyinhibit factor VIIa in the absence of factor Xa, and thus do not requireproduction of factor Xa via the intrinsic or extrinsic pathway. Moreparticularly, the present invention provides a novel, previously unknownKunitz-type inhibitor that shares amino acid sequence homology andoverall domain organization with tissue factor pathway inhibitor (TFPI).This novel Kunitz-type inhibitor has been designated TFPI-2.

Among the features of the present invention are isolated DNA moleculesencoding novel human Kunitz-type inhibitors. Such isolated molecules arethose that are separated from their natural environment and include cDNAand genomic clones. Isolated DNA molecules of the present invention areprovided free of other genes with which they are naturally associatedand may include naturally occurring 5′ and 3′ untranslated sequencesthat represent regulatory regions such as promoters and terminators. Theidentification of regulatory regions within the naturally occurring 5′and 3′ untranslated regions will be evident to one of ordinary skill inthe art (for review, see Dynan and Tijan, Nature 316: 774-778, 1985;Birnstiel et al., Cell 41: 349-359, 1985; Proudfoot, Trends in Biochem.Sci. 14: 105-110, 1989; and Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; whichare incorporated herein by reference).

The isolated DNA molecules of the present invention are useful inproducing recombinant human Kunitz-type inhibitors. Thus, the presentinvention provides the advantage that human Kunitz-type inhibitors areproduced in high quantities that may be readily purified using methodsknown in the art (see generally, Scopes, Protein Purification,Springer-Verlag, N.Y., 1982). Alternatively, the proteins of the presentinvention may be synthesized following conventional synthesis methodssuch as by the solid-phase synthesis such as the method of Barany andMerrifield (in The Peptides. Analysis, Synthesis, Biology Vol. 2, Grossand Meienhofer, eds, Academic Press, New York, pp. 1-284, 1980), bypartial solid-phase techniques, by fragment condensation or by classicalsolution addition.

Thus, an additional feature of the present invention is an isolatedhuman Kunitz-type inhibitor. Isolated proteins and peptides of thepresent invention are proteins of at least about 50% homogeneity, morepreferably of 70% to 80% homogeneity with a protein preparation of 95%to 99% or more homogeneity most preferred, particularly forpharmaceutical uses.

Kunitz-type inhibitor activity may be measured using the methodessentially described by Norris et al. (Biol. Chem. Hoppe-Seyler 371:37-42, 1990). Briefly, various fixed concentrations of the Kunitz-typeinhibitor are incubated in the presence of 0.24 μg/ml of porcine trypsin(Novo Nordisk A/S, Bagsvaerd, Denmark), 12.8 CU/l human plasmin (Kabi,Stockholm, Sweden) or 0.16 nkat/ml human plasma kallikrein (Kabi) in 100mM NaCl, 50 mM Tris HCl, 0.01% TWEEN 80 (Polyoxyethylenesorbitanmonoleate) (pH 7.4) at 25° C. After a 30 minute incubation, the residualenzymatic activity is measured by the cleavage of a solution containing0.6 mM of either of the chromogenic peptidyl nitroanilidetrypsin/plasmin substrates S2251 (D-Val-Leu-Lys-Nan; Kabi) or S2302(D-Pro-Phe-Arg-Nan; Kabi) in assay buffer. The samples are incubated for30 minutes after which the absorbance of each sample is measured at 405nm. An inhibition of enzyme activity is measured as a decrease inabsorbance at 405 nm or fluorescence Em at 460 nm. From the results, theapparent inhibition constant K_(i) is calculated.

The Kunitz-type inhibitors of the present invention may be used in thedisclosed methods for the treatment of, inter alia, deep veinthrombosis, disseminated intravascular coagulation, pulmonary embolismand for the prevention of thrombosis following surgery.

The present invention relates to novel human Kunitz-type inhibitorscomprising the amino acid sequence shown in SEQ ID NO:15, SEQ ID NO:2 orportions thereof and/or encoded by a DNA sequence comprising thenucleotide sequence of SEQ ID NO:14, SEQ ID NO:1 or portions thereof. Acomparison of the amino acid sequence SEQ ID NO:2 of TFPI-2 with otherKunitz-type inhibitors, more particularly with TFPI, showed that theprotein contains three putative Kunitz-type inhibitor domains. As willbe evident to one skilled in the art, each individual domain orcombinations of the domains may be prepared synthetically or byrecombinant DNA techniques for use in the present invention. Theputative Kunitz-type inhibitor domains comprise the amino acid sequenceshown SEQ ID NO:2 from cysteine, amino acid number 36 through cysteine,amino acid number 86; from cysteine, amino acid number 96 throughcysteine, amino acid number 149; and from cysteine, amino acid 158through cysteine amino acid 208. More particularly, the Kunitz-typeinhibitors of the present invention comprise the amino acid sequence ofSEQ ID NO:2 from cysteine, amino acid number 36 through cysteine, aminoacid number 86. Kunitz domains are defined by the location of the sixspecifically placed cysteine residues which are believed to formdisulfide bonds (See Laskowski and Kato, ibid. and Broze et al.,Biochemistry 29: 7539-7546, 1990). The first and sixth cysteine residuesdefine the boundaries of each Kunitz domain. Thus, in the case ofTFPI-2, the Kunitz domains are bounded by residues 36 and 86, 96 and149, 158 and 208 (numbered in accordance with SEQ ID NO:2). To providethe proper disulfide bond formation and protein conformation it isdesirable to include at least two amino acid residues flanking each ofthe cysteine residues defining the Kunitz domain. However, theidentities of these flanking residues are not critical. It is thuspossible to prepare variants of the individual Kunitz domains comprisingthe “core” Kunitz sequences described above, wherein the polypeptidecore is flanked on its amino and carboxyl termini by from two to four ormore amino acid residues other than cysteine residues. Furthermore, aswill be evident to one skilled in the art, amino-terminal and/orcarboxyl-terminal extensions of the Kunitz-type inhibitor may beprepared either synthetically or using recombinant DNA techniques andtested for inhibitor activity.

The DNA sequences encoding the proteins of the present invention wereunexpectedly identified during screening for a cDNA corresponding to thegenomic clone of a related but distinct Kunitz-type inhibitor using anantisense oligonucleotide probe complementary to a portion of theinhibitor coding sequence. Analysis of the cDNA clones revealed clonesthat encoded a unique, previously unknown Kunitz-type inhibitor,designated TFPI-2. The proteins of the present invention may be encodedby DNA sequences that are substantially similar to the DNA sequencedisclosed herein. As used within the context of the present invention,“substantially similar” DNA sequences encompass allelic variants andgenetically engineered or synthetic variants of the TFPI-2 gene thatcontain conservative amino acid substitutions and/or minor additions,substitutions or deletions of amino acids. DNA sequence variants alsoencompass degeneracies in the DNA code wherein host-preferred codons aresubstituted for the analogous codons in the human sequence. In addition,substantially similar DNA sequences are those that are capable ofhybridizing to the DNA sequences of the present invention under high orlow stringency (see Sambrook et al., ibid.) and those sequences that aredegenerate as a result of the genetic code, for example, to the aminoacid sequences of the present invention. Genetically engineered variantsmay be obtained by using oligonucleotide-directed site-specificmutagenesis, by use of restriction endonuclease digestion and adapterligation, or other methods well established in the literature (see forexample, Sambrook et al., ibid. and Smith et al., Genetic Engineering:Principles and Methods, Plenum Press, 1981; which are incorporatedherein by reference).

DNA molecules of the present invention may be isolated using standardcloning methods such as those described by Maniatis et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982; which isincorporated herein by reference), Sambrook et al. (Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; whichis incorporated herein by reference) or Mullis et al. (U.S. Pat. No.4,683,195; incorporated herein by reference). Alternatively, the codingsequences of the present invention may be synthesized using standardtechniques that are well known in the art, such as by synthesis on anautomated DNA synthesizer. As will be discussed in more detail below, anovel, previously unknown human Kunitz-type inhibitor was identified asa 1.0 kb cDNA insert and comprises the DNA sequence of SEQ ID NO:1. Inone embodiment of the invention, DNA sequences encoding the Kunitz-typeinhibitors of the present invention are obtained by PCR amplificationusing primers designed from SEQ ID NO:1 or its complement.

DNA molecules encoding TFPI-2 may also be obtained from non-humananimals such as dogs, rabbits, chicken, pigs, mice, rats and cows byscreening placental, liver or umbilical vein cell cDNA or genomiclibraries using the DNA sequences and methods disclosed herein.

DNA molecules of the present invention or portions thereof may be usedas probes, for example, to directly detect TFPI-2 sequences in cells.Such DNA molecules are generally synthetic oligonucleotides, but may begenerated from cloned cDNA or genomic sequences and will generallycomprise at least 12 nucleotides, more often from about 14 nucleotidesto about 25 or more nucleotides, sometimes 40 to 60 nucleotides, and insome instances a substantial portion or even the entire TFPI-2 gene orcDNA. The synthetic oligonucleotides of the present invention have atleast 85% identity to a corresponding TFPI-2 DNA sequence (SEQ ID NO:1)or its complement. For use as probes, the molecules are labeled toprovide a detectable signal, such as with an enzyme, biotin, aradionuclide, fluorophore, chemiluminescer, paramagnetic particle, etc.according to methods known in the art. Probes of the present inventionmay be used diagnostic methods to detect cellular metabolic disorderssuch as thrombolic disorders.

DNA molecules used within the present invention may be labeled and usedin a hybridization procedure similar to the Southern or dot blot. Aswill be understood by those skilled in the art, conditions that allowthe DNA molecules of the present invention to hybridize to the TFPI-2sequences may be determined by methods well known in the art and arereviewed, for example, by Sambrook et al. (Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; whichis incorporated herein by reference). Those skilled in the art will becapable of varying hybridization conditions (i.e. stringency ofhybridization) of the DNA molecules as appropriate for use in thevarious procedures by methods well known in the literature (see, forexample, Sambrook et al., ibid., pages 11.45-11.53). The higher thestringency of hybridization, the lower the number of mismatchedsequences detected. Alternatively, lower stringency will allow relatedsequences to be identified.

Alternatively, TFPI-2 protein sequence variants may be identified usingDNA molecules of the present invention and, for example, the polymerasechain reaction (PCR) (disclosed by Saiki et al., Science 239: 487, 1987;Mullis et al., U.S. Pat. No. 4,686,195; and Mullis et al., U.S. Pat. No.4,683,202) to amplify DNA sequences, which are subsequently detected bytheir characteristic size on agarose gels or which may be sequenced todetect sequence abnormalities.

DNA molecules encoding the Kunitz-type inhibitors of the presentinvention may be inserted into DNA constructs. As used within thecontext of the present invention, a DNA construct, also known as anexpression vector, is understood to refer to a DNA molecule, or a cloneof such a molecule, either single- or double-stranded, which has beenmodified through human intervention to contain segments of DNA combinedand juxtaposed in a manner that would not otherwise exist in nature. DNAconstructs of the present invention comprise a first DNA segmentencoding a Kunitz-type inhibitor operably linked to additional DNAsegments required for the expression of the first DNA segment. Withinthe context of the present invention, additional DNA segments willgenerally include promoters and transcription terminators, and mayfurther include enhancers and other elements. One or more selectablemarkers may also be included. DNA constructs useful for expressingcloned DNA segments in a variety of prokaryotic and eukaryotic hostcells can be prepared from readily available components or purchase fromcommercial suppliers.

In one embodiment the DNA sequence encodes a Kunitz-type inhibitorcomprising the amino acid sequence of SEQ ID NO:15 wherein each Xaa isindividually any amino acid except cysteine. In another embodiment theDNA sequence encodes a Kunitz-type inhibitor comprising the amino acidsequence of SEQ ID NO:2 from methionine, amino acid number 1 throughphenylalanine, amino acid number 235. In another embodiment, the firstDNA sequence encodes a Kunitz-type inhibitor comprising the amino acidsequence of SEQ ID NO:2 from glutamic acid, amino acid 34 to isoleucine,amino acid number 89. In another embodiment of the invention, theKunitz-type inhibitor comprises the amino acid sequence of SEQ ID NO:2from glutamic acid, amino acid number 34 to lysine, amino acid number152. In yet another embodiment of the invention, the Kunitz-typeinhibitor comprises the amino acid sequence of SEQ ID NO:2 from glutamicacid, amino acid number 34 to alanine, amino acid number 211.

DNA constructs may also contain DNA segments necessary to direct thesecretion of a polypeptide or protein of interest. Such DNA segments mayinclude at least one secretory signal sequence. Secretory signalsequences, also called leader sequences, prepro sequences and/or presequences, are amino acid sequences that act to direct the secretion ofmature polypeptides or proteins from a cell. Such sequences arecharacterized by a core of hydrophobic amino acids and are typically(but not exclusively) found at the amino termini of newly synthesizedproteins. Very often the secretory peptide is cleaved from the matureprotein during secretion. Such secretory peptides contain processingsites that allow cleavage of the secretory peptide from the matureprotein as it passes through the secretory pathway. A preferredprocessing site is a dibasic cleavage site, such as that recognized bythe Saccharomyces cerevisiae KEX2 gene. A particularly preferredprocessing site is a Lys-Arg processing site. Processing sites may beencoded within the secretory peptide or may be added to the peptide by,for example, in vitro mutagenesis.

Preferred secretory signals include the α factor signal sequence (preprosequence: Kurjan and Herskowitz, Cell 30: 933-943, 1982; Kurjan et al.,U.S. Pat. No. 4,546,082; Brake, EP 116,201), the PHO5 signal sequence(Beck et al., WO 86/00637), the BAR1 secretory signal sequence (MacKayet al., U.S. Pat. No. 4,613,572; MacKay, WO 87/002670), the SUC2 signalsequence (Carlsen et al., Molecular and Cellular Biology 3: 439-447,1983), the α-1-antitrypsin signal sequence (Kurachi et al., Proc. Natl.Acad. Sci. USA 78: 6826-6830, 1981), the α-2 plasmin inhibitor signalsequence (Tone et al., J. Biochem. (Tokyo) 102: 1033-1042, 1987) and thetissue plasminogen activator signal sequence (Pennica et al., Nature301: 214-221, 1983). Alternately, a secretory signal sequence may besynthesized according to the rules established, for example, by vonHeinje (European Journal of Biochemistry 133: 17-21, 1983; Journal ofMolecular Biology 184: 99-105, 1985; Nucleic Acids Research 14:4683-4690, 1986). A particularly preferred signal sequence is thesynthetic signal LaC212 spx (1-47)—ERLE described in WO 90/10075, whichis incorporated by reference herein in its entirety.

Secretory signal sequences may be used singly or may be combined. Forexample, a first secretory signal sequence may be used in combinationwith a sequence encoding the third domain of barrier (described in U.S.Pat. No. 5,037,243, which is incorporated by reference herein in itsentirety). The third domain of barrier may be positioned in properreading frame 3′ of the DNA segment of interest or 5′ to the DNA segmentand in proper reading frame with both the secretory signal sequence anda DNA segment of interest.

The choice of suitable promoters, terminators and secretory signals iswell within the level of ordinary skill in the art. Methods forexpressing cloned genes in Saccharomyces cerevisiae are generally knownin the art (see, “Gene Expression Technology,” Methods in Enzymology,Vol. 185, Goeddel (ed.), Academic Press, San Diego, Calif., 1990 and“Guide to Yeast Genetics and Molecular Biology,” Methods in Enzymology,Guthrie and Fink (eds.), Academic Press, San Diego, Calif., 1991; whichare incorporated herein by reference) . Proteins of the presentinvention can also be expressed in filamentous fungi, for example,strains of the fungi Aspergillus (McKnight et al., U.S. Pat. No.4,935,349, which is incorporated herein by reference) . Expression ofcloned genes in cultured mammalian cells and in E. coli, for example, isdiscussed in detail in Sambrook et al. (Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., 1989; which isincorporated herein by reference). As would be evident to one skilled inthe art, one could express the proteins of the instant invention inother host cells such as avian, insect and plant cells using regulatorysequences, vectors and methods well established in the literature.

In yeast, suitable yeast vectors for use in the present inventioninclude YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039,1978), YEp13 (Broach et al., Gene 8: 121-133, 1979), POT vectors(Kawasaki et al, U.S. Pat. No. 4,931,373, which is incorporated byreference herein), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978)and derivatives thereof. Preferred promoters for use in yeast includepromoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem.255: 12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or alcoholdehydrogenase genes (Young et al., in Genetic Engineering ofMicroorganisms for Chemicals, Hollaender et al., (eds.), p. 355, Plenum,N.Y., 1982; Ammerer, Meth. Enzymol. 101: 192-201, 1983). In this regard,particularly preferred promoters are the TPI1 promoter (Kawasaki, U.S.Pat. No. 4,599,311, 1986) and the ADH2-4^(c) promoter (Russell et al.,Nature 304: 652-654, 1983; Irani and Kilgore, U.S. patent applicationSer. No. 07/784,653, CA 1,304,020 and EP 284 044, which are incorporatedherein by reference). The expression units may also include atranscriptional terminator. A preferred transcriptional terminator isthe TPI1 terminator (Alber and Kawasaki, ibid.).

Host cells containing DNA constructs of the present invention are thencultured to produce the Kunitz-type inhibitors. The cells are culturedaccording to standard methods in a culture medium containing nutrientsrequired for growth of the particular host cells. A variety of suitablemedia are known in the art and generally include a carbon source, anitrogen source, essential amino acids, vitamins, minerals and growthfactors. The growth medium will generally select for cells containingthe DNA construct by, for example, drug selection or deficiency in anessential nutrient which is complemented by a selectable marker on theDNA construct or co-transfected with the DNA construct.

Yeast cells, for example, are preferably cultured in a chemicallydefined medium, comprising a non-amino acid nitrogen source, inorganicsalts, vitamins and essential amino acid supplements. The pH of themedium is preferably maintained at a pH greater than 2 and less than 8,preferably at pH 6.5. Methods for maintaining a stable pH includebuffering and constant pH control, preferably through the addition ofsodium hydroxide. Preferred buffering agents include succinic acid andBis-Tris (Sigma Chemical Co., St. Louis, Mo.). Yeast cells having adefect in a gene required for asparagine-linked glycosylation arepreferably grown in a medium containing an osmotic stabilizer. Apreferred osmotic stabilizer is sorbitol supplemented into the medium ata concentration between 0.1 M and 1.5 M, preferably at 0.5 M or 1.0 M.Cultured mammalian cells are generally cultured in commerciallyavailable serum-containing or serum-free media. Selection of a mediumappropriate for the particular host cell used is within the level ofordinary skill in the art.

Within one embodiment of the invention, the proteins of the presentinvention are expressed in mammalian cells. Methods for introducingexogenous DNA into mammalian host cells include calciumphosphate-mediated transfection (Wigler et al., Cell 14:725, 1978;Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Vander Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.1:841-845, 1982) and DEAE-dextran mediated transfection (Ausubel et al.,eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc.,New York, 1987), which are incorporated herein by reference. Cationiclipid transfection using commerically available reagents including theBoehringer Mannheim Transfection-Reagent(N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl ammoniummethylsulfate;Boehringer Mannheim, Indianapolis, Ind.) or LIPOFECTIN reagent(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride anddioeleoyl phosphatidylethanolamine; GIBCO-BRL, Gaithersburg, Md.) usingthe manufacturer-supplied directions, may also be used. The productionof recombinant proteins in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134, which are incorporated herein byreference. Preferred cultured mammalian cells include the COS-1 (ATCCNo. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK570 (ATCC No. CRL 10314) and 293 (ATCC No. CRL 1573; Graham et al., J.Gen. Virol. 36:59-72, 1977) cell lines. Additional suitable cell linesare known in the art and available from public depositories such as theAmerican Type Culture Collection, Rockville, Md.

The recombinant Kunitz-type inhibitors expressed using the methodsdescribed herein are isolated and purified by conventional procedures,including separating the cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulfate,purification by a variety of chromatographic procedures, e.g. ionexchange chromatography or affinity chromatography, or the like. Methodsof protein purification are known in the art (see generally, Scopes, R.,Protein Purification, Springer-Verlag, N.Y. (1982), which isincorporated herein by reference) and may be applied to the purificationof the recombinant proteins of the present invention.

The Kunitz-type inhibitors of the present invention may be purifiedusing the ability of the inhibitor to bind to trypsin. Briefly, a totalof approximately 1 liter fermentation supernatant is adjusted to pH 8.0by the addition of solid Tris-HCl to a final concentration of 50 mM andtitration with 4 M NaOH. After filtration to remove any cellular debris,the supernatant is applied to a column of bovine trypsin adsorbed toCNBr-activated Sepharose (350 mg bovine trypsin per 35 ml gel). Thecolumn is washed sequentially with 150 ml 0.1 M Tris-HCl (pH 8.0), 0.5 MNaCl, then 150 ml 0.01 M Tris-HCl (pH 8.0) before the bound material iseluted with 200 ml 0.2 M glycine-HCl (pH 3.0). Fractions of 10 ml arecollected and analyzed by reverse phase HPLC. Protein-containingfractions are combined.

The pooled material is applied to a preparative reverse phase HPLCcolumn, (Vydac, The Separations Group, Hesperia, CA or the like)equilibrated with 5% B (0.7% TFA in acetonitrile) and 95% A (0.1% TFA inH₂O). The flow rate is maintained at 4 ml/min. Following application ofsample, the column is washed with 5% B until a baseline at 214 nm isachieved. Gradient elution with fraction collection is performed from 5to 85% B over 80 min. Fractions containing UV-absorbing material areanalyzed by reverse phase HPLC (Vydac) and combined to give pools ofchromatographically pure material. Solvent is removed from the pooledfractions by vacuum centrifugation. The concentration and total yield ofinhibitor in the major pools is estimated by reverse phase HPLC analysisand by comparison to an aprotinin standard. The final preparations arecharacterized by electronspray mass spectroscopy (SCIEX API III) or thelike.

In cases where proteolytic cleavage of the Kunitz inhibitor is apotential problem, the Kunitz inhibitors of the present invention mayalso be purified using the method essentially described by Norris et al.(Biol. Chem. Hoppe-Seyler 371: 37-42, 1990, which is incorporated byreference herein in its entirety). Briefly, selected transformants aregrown in 10 liters of YEPD for approximately 40 hours at 30° C. until anOD₆₀₀ of approximately 25 has been reached. The culture is centrifuged,and the supernatant is decanted. A 300 ml-1000 ml aliquot of supernatantis adjusted to pH 2.3 and applied to a column holding 8 ml of beadedagarose matrix such as S-Sepharaose (Pharmacia-LKB Biotechnology AS,Alleroed, Denmark) or the like that has been previously equilibratedwith 20 mM Bicine, pH 8.7 (Sigma Chemical Co., St. Louis, Mo.). Afterthe column has been extensively washed with 20 mM Bicine, pH 8.7, theKunitz-type inhibitor is eluted with 30 ml of 20 mM Bicine, pH 8.7containing 1 M NaCl. The eluted material is desalted by application to aSephadex G-25 column (a beaded dextran matrix, Pharmacia-LKBBiotechnology AS, Alleroed, Denmark; 2.5×30 cm) or the like that hasbeen equilibrated with 20 mM NH₄HCO₃, pH 7.8. The Kunitz-type inhibitoris eluted with 20 mM NH₄HCO₃, pH 7.8.

The Kunitz-type inhibitors are further purified and concentrated bychromatography on a column containing a cation exchanger with chargedsulfonic groups coupled to a beaded hydrophylic resin such as a MONO Scolumn (Pharmacia-LKB Biotechnology AS, Alleroed, Denmark; 0.5×5 cm) orthe like equilibrated with 20 mM Bicine, pH 8.7. After washing with theequilibration buffer at 2 ml/min for 10 minutes, gradient elution of theKunitz-type inhibitor is carried out over twelve minutes at 1 ml/minfrom 0-0.6 M NaCl in the equilibration buffer. Peak samples are pooled,and the Kunitz-type inhibitor is purified using reverse phase HPLC on aVydac 214TP510 column (Mikro-lab, Aarhus, Denmark; 1.0×25 cm) or thelike with a gradient elution at 4 ml/min from 5% A (0.1% trifluoroaceticacid (TFA) in water) to 45% B (0.7% TFA in acetonitrile) in 20 minutes.The purified product in lyophilized in water, and inhibitor activity ismeasured.

Alternatively, TFPI-2 may be purified from conditioned medium bysequential chromatography using heparin agarose, an anion exchanger withquaternary amine groups crosslinked to a beaded hydrophylic resin suchas MONO Q (Pharmacia) or the like, a cation exchanger with chargedsulfonic groups coupled to a beaded hydrophylic resin such as MONO S(Pharmacia) or the like and cross-linked agarose gel filtration matrixhaving different porosities for the separation of proteins from 1×10³ to3×10⁵ MW such as SUPEROSE 12 (Pharmacia) or the like. Briefly,conditioned serum-free media, adjusted to pH 7.5 with 1 N NaOH andfiltered through a 0.22-μm filter, is applied to a heparin sepharosecolumn (Pharmacia Biotech Inc., Piscataway, N.J.) or the like that hasbeen equilibrated at 4° C. with Buffer A (50 mM Tris-HCl (pH 7.5), 10%glycerol). The filtered media is applied at a flow rate of 3 ml/min. Thecolumn is washed with Buffer A containing 0.2 M NaCl. TFPI-2 activity,as judged by its ability to inhibit trypsin (Example 4A), is eluted fromthe column with Buffer A containing 1 M NaCl. The eluent from theheparin sepharose column is dialyzed at 4° C. against 25 mM Tris-HCl (pH7.5), 10% glycerol. The retentate is subjected to FPLC (PharmaciaBiotech Inc.) on a 5×50 mm column containing an anion exchanger withquaternary amine groups crosslinked to a beaded hydrophylic resin suchas a MONO Q (MONO Q HR 5/5; Pharmacia Biotech Inc., Piscataway, N.J.) orthe like that had been equilibrated with 25 mM Tris-HCl (pH 7.5), 10%glycerol at room temperature. TFPI-2 is eluted from the column in alinear NaCl gradient (from 0-0.5 M NaCl) at a flow rate of 1 ml/min. TheTFPI-2 fractions are pooled and dialyzed against 25 mM sodium citrate(pH 5.0), 10% glycerol. The retentate is then subjected to FPLC at roomtemperature on a cation exchanger with charged sulfonic groups coupledto a beaded hydrophylic resin such as MONO S (MONO S HR 5/5, PharmaciaBiotech Inc.) or the like at a flow rate of 0.5 ml/min. TFPI-2 activityis eluted from the MONO S column with a gradient elution from 25 mMsodium citrate (pH 5.0), 10% glycerol to 25 mM Tris-HCl (pH 7.5), 10%glycerol, 1 M NaCl. Fractions containing TFPI-2 activity are pooled andconcentrated to approximately 1 ml by ultrafiltration. The concentratedsamples are subjected to FPLC across a cross-linked agarose gelfiltration matrix having a porosity suitable for the separation ofproteins from 1×10³ to 3×10⁵ MW such as SUPEROSE 12 (Pharmacia BiotechInc., Piscataway, N.J.) or the like at room temperature in 50 mMTris-HCl (pH 7.5), 100 mM NaCl. Fractions eluted from the FPLC withTFPI-2 activity were subjected to SDS-PAGE, and pure fractions arepooled and stored at −80° C.

The present invention also relates to a pharmaceutical compositioncomprising a Kunitz-type inhibitor of the present invention togetherwith a pharmaceutically acceptable carrier or vehicle. In thecomposition of the invention, the Kunitz-type inhibitor may beformulated by any of the established methods of formulatingpharmaceutical compositions, e.g. as described in Remington'sPharmaceutical Sciences, 1985. The composition may typically be in aform suited for systemic injection or infusion and may, as such, beformulated with sterile water or an isotonic saline or glucose solution.

Kunitz-type inhibitors of the present invention are thereforecontemplated to be advantageous for use in therapeutic applications forwhich tissue factor pathway inhibitor are useful. Such applicationsinclude disseminated intravascular coagulation, deep vein thrombosis,pulmonary embolism and in the prevention of thrombosis followingsurgery. As will be evident to one skilled in the art, the Kunitz-typeinhibitors of the present invention may be combined with othertherapeutic agents to augment the antithrombotic or anticoagulantactivity of such agents. TFPI-2 may, for example, be used in conjunctionwith tissue plasminogen activator in thrombolytic therapy. The use ofthe Kunitz-type inhibitors of the present invention is indicated as aresult of their ability to inhibit factor VIIa/tissue factor complex.

Thus, the Kunitz-type inhibitors of the present invention may be usedwithin methods for inhibiting blood coagulation in mammals. Such methodswill generally include administering the Kunitz-type inhibitor in anamount sufficient to inhibit blood coagulation. Such amounts can varyaccording to the severity of the condition being treated and may rangefrom approximately 10 μg/kg to 10 mg/kg body weight. Preferably theamount of the Kunitz-type inhibitor administered will be within therange of 100 μg/kg and 5 mg/kg with a range of 100 μg/kg and 1 mg/kg asthe most preferable range.

Apart from the pharmaceutical use indicated above, the Kunitz-typeinhibitors as specified above may be used to isolate useful naturalsubstances, e.g. proteases or receptors from human material, which binddirectly or indirectly to the Kunitz-type inhibitor, for instance byscreening assays or by affinity chromatography.

Within one aspect of the present invention, Kunitz-type inhibitors,including derivatives thereof, as well as portions or fragments of theseproteins, are utilized to prepare antibodies which specifically bind tothe Kunitz-type inhibitors. As used herein, the term “antibodies”includes polyclonal antibodies, monoclonal antibodies, antigen-bindingfragments thereof such as F(ab′)₂ and Fab fragments, as well asrecombinantly produced binding partners. These binding partnersincorporate the variable regions from a gene which encodes aspecifically binding monoclonal antibody. Antibodies are defined to bespecifically binding if they bind to the Kunitz-type inhibitor with aK_(a) of greater than or equal to 10⁷/M. The affinity of a monoclonalantibody or binding partner may be readily determined by one of ordinaryskill in the art (see, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).Isolated antibodies are those antibodies that are substantially free ofother blood.

Methods for preparing polyclonal and monoclonal antibodies have beenwell described in the literature (see for example, Sambrook et al.,ibid.; Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:Techniques and Applications, CRE Press, Inc., Boca Raton, Fla., 1982).As would be evident to one of ordinary skill in the art, polyclonalantibodies may be generated from a variety of warm-blooded animals suchas horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats.The immunogenicity of the Kunitz-type inhibitor may be increased throughthe use of an adjuvant such as Freund's complete or incomplete adjuvant.A variety of assays known to those skilled in the art may be utilized todetect antibodies which specifically bind to a Kunitz-type inhibitor.Exemplary assays are described in detail in Antibodies: A LaboratoryManual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press,1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations,enzyme-linked immuno-sorbent assays, dot blot assays, inhibition orcompetition assays, and sandwich assays.

Additional techniques for the preparation of monoclonal antibodies mayutilized to construct and express recombinant monoclonal antibodies.Briefly, mRNA is isolated from a B cell population and utilized tocreate heavy and light chain immunoglobulin cDNA expression libraries ina suitable vector such as the XIMMUNOZAP(H) and XIMMUNOZAP(L) vectors,which may be obtained from Stratocyte (La Jolla, Calif.). These vectorsare then screened individually or are co-expressed to form Fab fragmentsor antibodies (Huse et al., Science 246: 1275-1281, 1989; Sastry et al.,Proc. Natl. Acad. Sci. USA 86: 5728-5732, 1989). Positive plaques aresubsequently converted to a non-lytic plasmid which allows high levelexpression of monoclonal antibody fragments in E. coli.

Binding partners such as those described above may also be constructedutilizing recombinant DNA techniques to incorporate the variable regionsof a gene which encodes a specifically binding antibody. Theconstruction of these proteins may be readily accomplished by one ofordinary skill in the art (see for example, Larrick et al.,Biotechnology 7: 934-938, 1989; Reichmann et al., Nature 322: 323-327,1988 and Roberts et al. Nature 328: 731-734, 1987). Once suitableantibodies or binding partners have been obtained, they may be isolatedor purified by many techniques well described in the literature (see forexample, Antibodies: A Laboratory Manual, ibid.). Suitable techniquesinclude protein or peptide affinity columns, HPLC or RP-HPLC,purification on protein A or protein G columns or any combination ofthese techniques. Within the context of the present invention, the term“isolated” as used to define antibodies or binding partners means“substantially free of other blood components.”

Antibodies and binding partners of the present invention may be used ina variety of ways. The tissue distribution of the Kunitz-type inhibitor,for example, may be determined by incubating tissue slices with alabeled monoclonal antibody which specifically binds to the Kunitz-typeinhibitor, followed by detection of the presence of the bound antibody.Labels suitable for use within the present invention are well known inthe art and include, among others, fluorescein, isothiocyanate,phycoerythrin, horseradish peroxidase, and colloidal gold. Theantibodies of the present invention may also be used for thepurification of the Kunitz-type inhibitors of the present invention. Thecoupling of antibodies to solid supports and their use in purificationof proteins is well known in the literature (see for example, Methods inMolecular Biology, Vol. 1, Walker (Ed.), Humana Press, New Jersey, 1984,which is incorporated by reference herein in its entirety).

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLES

Restriction endonucleases and other DNA modification enzymes (e.g., T4polynucleotide kinase, calf alkaline phosphatase, DNA polymerase I(Klenow fragment), T4 polynucleotide ligase) were obtained from GIBCOBRL Life Technologies, Inc (GIBCO BRL) and New England Biolabs and wereused as directed by the manufacturer, unless otherwise noted.

Oligonucleotides were synthesized on an Applied Biosystems Model 380ADNA synthesizer and purified by polyacrylamide gel electrophoresis ondenaturing gels. E. coli cells were transformed as described by Maniatiset al. (Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, 1982) or Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2 ed., Cold Spring Harbor, N.Y., 1989). Radiolabeled probes andhybridization solutions were prepared essentially as described bySambrook et al. (Molecular Cloning: A Laboratory Manual, 2 ed., ColdSpring Harbor, N.Y., 1989; which is incorporated by reference herein inits entirety).

Example 1 Cloning of A Novel Human Kunitz Inhibitor cDNA

Poly(A)⁺ RNAs from a variety of human tissue sources were screened usingan antisense 30-mer oligonucleotide (ZC4792; SEQ ID NO:3) A blot ofhuman poly(A)⁺ RNA from heart, brain, placenta, liver, lung, skeletalmuscle, kidney and pancreas (HUMAN MTN BLOT) was obtained from ClontechLaboratories, Inc. (Palo Alto, Calif.). The blot was prehybridized in aprehybridization solution (5×SSPE (Table 1), 2×Denhardt's (Table 1),0.5% sodium dodecylsulfate (SDS), 100 μg/ml of sonicated salmon spermDNA) for four hours at 55° C. After prehybridization, theprehybridization solution was removed and replaced with prehybridizationsolution containing 4.7×10⁶ cpm/ml of ³²P-labeled ZC4792 (SEQ ID NO:3).After an overnight incubation at 55° C. the hybridization solution wasremoved, and the blot was washed once in 2×SSC (Table 1), 0.05% SDS atroom temperature for 20 minutes followed by a wash in 2×SSC (Table 1),0.1% SDS for 20 minutes at 55° C. The blot was exposed to film for twoand a half hours. The resulting autoradiograph showed a number of bandsin most of the lanes, indicating the presence of related sequences inmost of the tissues represented in the blot. The blot was washed at ahigher stringency in 2×SSC (Table 1) at a temperature between 60° C. and65° C. for 30 minutes, after which the blot was exposed to filmovernight. The second autoradiograph showed the presence of a 1.6 kbband for placenta and liver and an apparently smaller band ofapproximately 1.2 kb in the pancreas. TABLE 1 20 × SSPE 175.3 g NaCl 27.6 g NaH₂PO₄.H₂O  7.4 g EDTA Dissolve the solids in 800 ml ofdistilled water. Adjust the pH to 7.4 with NaOH (approximately 6.5 ml ofa 10 N solution). Adjust the volume to 1 liter with distilled water.Sterilize by autoclaving. 50 × Denhardt's 5 g Ficoll 5 gpolyvinylpyrrolidone 5 g bovine serum albumin (Fraction V) Dissolve thesolids into a final volume of 500 ml. Filter the solution to sterilizeand store at −20° C. 20 × SSC 175.3 g NaCl  88.2 g sodium citrateDissolve the solids in 800 ml of distilled water. Adjust the pH to 7.0by a drop-wise addition of 10 N NaOH. Adjust the volume to 1 liter withdistilled water. Sterilize by autoclaving. Prehybridization Solution #15 × SSPE 5 × Denhardt's 0.5% SDS 100 μg/ml sheared salmon sperm DNAPrehybridization Solution #2 5 × SSC 5 × Denhardt's 0.1% SDS 100 μg/mlsheared salmon sperm DNA Growth Medium Dulbecco's Modified Eagle'sMedium (DMEM) containing 5% fetal bovine serum, 2 mM L- glutamate, 1 ×PSN (50 μg/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml neomycin;GIBCO BRL), 10 μM methotrexate. Serum-free Medium  500 ml Dulbecco'sModified Eagle's Medium (DMEM) 0.29 mg/ml L-glutamine   10 mg/Ltransferrin   5 mg/L fetuin (Aldrich, Milwaukee, WI)   5 mg/L insulin(GIBCO BRL, Grand Island, NY)   2 μg/L selenium (Aldrich, Milwaukee, WI)In addition to the above ingredients, the medium was supplemented with10 μM methotrexate, 25-50 mM HEPES BUFFER SOLUTION (N-2-Hydroxyethylpiperazine-N′-2-Ethane Sulfonic Acid (pH 7.2); JRHBiosciences, Lenxa, KS) and 1 × PSN (GIBCO BRL). Phosphate BufferedSaline (PBS)   8 g sodium chloride 0.2 g potassium chloride   1 g sodiumphosphate   2 g potassium phosphate Dissolve solids in distilled water.Bring volume to 1 liter. Autoclave to sterilize.

To obtain a cDNA encoding a human placental protease inhibitor from theKunitz family, a human placenta cDNA library in λgt11 (ClontechLaboratories, Inc., Palo Alto, Calif.) was screened using theradio-labeled ZC4792 (SEQ ID NO:3) essentially as described above. Thelibrary was titered, and 2×10⁵ pfu/plate were plated on a total oftwelve plates to obtain 2.4 million independent plaques. Duplicateplaque lifts were prepared using ICN BIOTRANS nylon membranes (ICN,Irvine, Calif.). The membranes were prewashed in 5×SSC (Table 1), 0.5%SDS at 50° C. for one hour followed by an overnight prehybridization at55° C. in prehybridization solution #1 (Table 1). The prehybridizationsolution was removed and replaced with fresh prehybridization solution#1 (Table 1) containing 7.2×10⁷ cpm of ZC4792 probe (SEQ ID NO:3).Hybridization was carried out under the same conditions as theprehybridization. The hybridization solution was removed, and the blotswere washed at 60° C. in 2×SSC (Table 1), 0.1% SDS. Fourteen positiveplaques were identified and plaque purified using radio-labeled ZC4792(SEQ ID NO:3).

Tertiary filters from the plaque purifications of the fourteen cloneswere probed with a specific fragment from ZGKI13, a clone containing theamyloid precursor protein homologue coding sequence (deposited with theAmerican Type Culture Collection, 12301 ParkLawn Dr., Rockville, Md. onOct. 14, 1992, as an E. coli transformant under accession number ATCC69090) to identify and eliminate clones having homology with the amyloidprecursor protein homologue. A random-primed 880 bp Pst I-Xho I fragmentof ZGKI13 was used as a probe. The filters were hybridized overnight at65° C. in prehybridization solution #2 containing 2×10⁶ cpm/ml of thelabeled probe. After hybridization, the solution was removed, and thefilters were washed at 65° C. in 0.2×SSC (Table 1), 0.1% SDS. Four ofthe fourteen plaques were shown to encode the ZGKI13 amyloid proteinprecursor. These four clones were discarded.

Double-stranded DNA was prepared from one of the ten remaining purifiedphage clones, designated J-2-11. The plasmid DNA was digested with EcoRI to isolate the approximately 1 kb cDNA insert. The Eco RI fragmentwas subcloned into Eco RI-linearized pUC19. Sequence analysis of thecloned fragment demonstrated three regions of the clone that showedstrong homology to the Kunitz family of protease inhibitors. Thetertiary filters of the nine remaining phage clones (described above)were screened determined with a labeled probe specific to the J-2-11clone. The tertiary filters were hybridized overnight at 55° C. inprehybridization solution #2 (Table 1) containing 2×10⁶ cpm/ml of thekinased oligonucleotide ZC6281 probe (SEQ ID NO:4). After hybridization,the probe was removed, and the filters were washed at 60° C. in 2×SSC(Table 1), 0.1% SDS. Autoradiography of the filters showed that all ninecandidate clones contained sequences homologous to J-2-11. One clone wasselected and designated J-2-11/pUC19.

Plasmid J-2-11/pUC19 was deposited as an E. coli transformant on Sep.17, 1993 with the American Type Culture Collection (12301 Parklawn Dr.,Rockville, Md.) under accession number 69425. Plasmid J-2-11/pUC19 wasshown to contain the sequence shown in SEQ ID NO:1. Analysis of thesequence showed a 5′ noncoding region of 36 nucleotides, an open readingframe of 705 nucleotides encoding 235 amino acids, and a 235 nucleotide3′ noncoding region. A comparison of the deduced amino acid sequence((SEQ ID NO:1 and SEQ ID NO:2) with other Kunitz-type inhibitors showedamino acid homology and domain structure similarities with TFPI.

A blot of poly(A)⁺ mRNA from human tissues (Clontech Multiple TissueNorthern Blot) was screened using a ³²P-end-labeled oligonucleotidecorresponding to TFPI-2 sequences (ZC6281; SEQ ID NO:4) to determine thetissue distribution of the transcript. The blot was prehybridized in aprehybridization solution containing 5×SSPE (Table 1), 2×Denhardt's(Table 1), 0.5% SDS, 100 μg/ml salmon sperm DNA at 55° C. for severalhours. After prehybridization, the solution was removed, and the blotwas hybridized overnight at 55° C. in fresh prehybridization solutioncontaining the kinase ZC6281 (SEQ ID NO:4). The blot was washed at 65°C. in 0.2×SSC (Table 1), 0.1% SDS and exposed to film. Analysis of theautoradiograph indicated that TFPI-2 is transcribed in the placenta andliver. Subsequent northern analysis demonstrated the presence of aTFPI-2 transcript in human umbilical vein endothelial cells One majortranscript is apparent at 1.4 kb with a possible minor transcript at ˜2kb. Based on the size of the longest TFPI-2 clones, it is possible thatthe clone represents an incomplete transcript that is missing some ofthe 3′ non-coding sequence since no polyadenylation sequence is seen.The Eco RI site at the 3′ end appears to be an internal site as nolinker sequence is seen at this end. Therefore, the mRNA size wouldpredict an additional 400 bp of 3′ (or 5′) noncoding sequence in afull-length transcript.

Example 2 Expression of A Novel Human Kunitz-Type Inhibitor in CulturedMammalian Cells

The novel human Kunitz-type inhibitor encoded by clone J-2-11 wasexpressed in the mammalian expression vector Zem229R. The vector Zem229Rwas deposited on Sep. 28, 1993 with the American Type Culture Collection(12301 Parklawn Dr. Rockville, Md. 20852) as an E. coli transformantunder accession number 69447. The approximately 1 kb Eco RI fragmentfrom J-2-11/pUC19 was ligated into Zem229R that had been linearized bydigestion with Eco RI. Transformants were screened for plasmidscontaining the insert in the proper orientation relative to thepromoter. A positive clone was identified, and plasmid DNA was prepared.The plasmid DNA was used to transfect BHK570 cells using calciumphosphate-mediated transfection (Wigler et al., Cell 14:725, 1978;Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Vander Eb, Virology 52:456, 1973). BHK 570 cells were deposited with theAmerican Type Culture Collection (ATCC; 12301 Parklawn Dr., Rockville,Md., 20852, USA) on Dec. 20, 1989 under accession number CRL 10314.Transfected cells were initially selected in the presence of mediumcontaining 1 μM of methotrexate followed by more stringent selection inmedium containing 10 μM methotrexate. Following selection in 10 μMmethotrexate, randomly selected clones were grown to confluency in6-well dishes in Growth Medium (Table 1). After reaching confluency, thespent medium was decanted, and the cells were washed with PhosphateBuffered Saline (PBS; Table 1) to remove any remaining serum. Serum-freemedium (Table 1) was added to the cells, and the cells were grown for24-48 hours. The conditioned media was collected and assayed for trypsininhibitor activity using the assay method detailed in Example 4A.

A clone having the highest level of trypsin inhibitor activity wasselected for large-scale culture. Cells from the clone were expanded andseeded into either a small or large cell-factory and were grown toconfluency in growth medium (Table 1) containing 10 mg/L aprotinin (NovoNordisk A/S, Bagsvaerd, Denmark). After reaching confluency, the mediawas removed, the cells were washed with PBS and serum-free medium(Table 1) containing 10 mg/L aprotinin was added. Media was collectedevery 2-4 days and stored at −20° C.

Example 3 Expression of Kunitz-Type Inhibitor Domains in the YeastSaccharomyces cerevisiae

A. Expression of a Kunitz-Type Inhibitor Domain of the TFPI-2 ComprisingAmino Acids 34 through 89 of SEQ ID NO:2

The Kunitz-type inhibitor domain encoded in plasmid pJ-2-11/pUC19 andcomprising the amino acid sequence of SEQ ID NO:2 from glutamic acid,amino acid 34 through isoleucine, amino acid number 89 is expressed in astrain of the yeast Saccharomyces cerevisiae from a PCR-generatedsequence. The DNA sequence encoding the Kunitz-type inhibitor domain isamplified from pJ-2-11/pUC19. Synthetic oligonucleotide primers M-2161and M-2177 (SEQ ID NOS:5 and 6, respectively) are designed as PCRamplification primers. Synthetic oligonucleotide M-2177 is complementaryto nucleotides 288-305 of SEQ ID NO:1, and in addition carries a 5′extension containing a translation stop codon followed by an Xba I site.Oligonucleotide M-2161 contains a sequence that is identical tonucleotides 215-235 of the synthetic leader sequence shown in SEQ IDNO:7 followed by nucleotides 138-154 of SEQ ID NO:1. A PCR reaction isperformed in a 100 μl final volume using 1 μg plasmid pJ-2-11/pUC19, 100pmole each of oligonucleotides M-2161 and M-2177 (SEQ ID NOS:5 and 6,respectively), and the reagents provided in the GENEAMP kit (PerkinElmer Cetus, Norwalk, Conn.) according to the manufacturer'sinstructions. The reaction is amplified for nineteen cycles (20 secondsat 94° C., 20 seconds at 50° C., and 30 seconds at 72° C.) followed by aten minute incubation at 72° C. A 205 bp fragment is isolated by agarosegel electrophoresis.

A DNA sequence encoding the synthetic signal sequence (SEQ ID NO:7) isobtained by PCR amplification of a fragment from plasmid pKFN-1000.Plasmid pKFN-1000 is a derivative of plasmid pTZ19R (Mead et al., Prot.Engin. 1: 67-74, 1986) containing a DNA sequence encoding a syntheticyeast signal leader peptide. Plasmid pKFN-1000 is described in WO90/10075, which is incorporated by reference herein in its entirety. TheDNA sequence of the 235 base pairs downstream from the Eco RI site ofplasmid pKFN-1000 and the encoded amino acid sequence is shown in SEQ IDNOS: 7 and 8. A 0.7 kb Pvu II fragment of plasmid pKFN-1000 is used as atemplate. Synthetic oligonucleotide NOR-1478 (SEQ ID NO:9) is identicalto a sequence just upstream of the Eco RI site (nucleotides to 1-6 ofSEQ ID NO:7). Synthetic oligonucleotide NOR-2523 (SEQ ID NO:10) iscomplementary to nucleotides 215-235 of the coding sequence in SEQ IDNO:7. A PCR reaction is performed in a 100 μl final volume using 0.1 μgof the 0.7 kb Pvu II fragment, 100 pmoles each of oligonucleotidesNOR-1478 and NOR-2523 (SEQ ID NOS: 9 and 10, respectively) and reagentsfrom the GENEAMP commercial kit (Perkin Elmer Cetus) according to themanufacturer's instructions. The PCR reaction is amplified as describedabove. A 257 bp PCR product is isolated by agarose gel electrophoresis.

A DNA sequence encoding the complete synthetic signal sequenceoperatively linked to the Kunitz-type inhibitor domain sequence isobtained by amplifying the two PCR fragments described above. A PCRreaction is performed as described above using 100 pmoles each ofprimers NOR-1478 (SEQ ID NO:9) and M-2177 (SEQ ID NO:6) and 0.1 μg ofeach of the two PCR fragments described above. The PCR reaction isamplified for sixteen cycles (1 minute at 94° C., 2 minutes at 50° C., 3minutes at 71° C.) followed by a ten minute incubation at 72° C. A 437bp fragment is purified by agarose gel electrophoresis. The fragment isthen digested with Eco RI and Xba I, and the resulting 408 bp fragmentis ligated with plasmid pTZ19R, which had been linearized by digestionwith Eco RI and Xba I. The ligation mixture is transformed intocompetent restriction minus, modification plus E. coli strain, andtransformants were selected in the presence of ampicillin. Plasmid DNAsprepared from selected transformants are sequenced, and a plasmidcontaining the DNA sequence of the synthetic yeast signal sequence fusedto the Kunitz-type inhibitor domain is identified.

The Eco RI-Xba I fragment encoding the secretory signal-Kunitz-typeinhibitor domain is then isolated and subcloned into plasmid pMT-636.Plasmid pMT-636 was derived from the shuttle vector pCPOT (Plasmid pCPOTwas deposited on May 9, 1984 with the American Type Culture Collection;12301 Parklawn Dr., Rockville, Md.; under Accession No. 39685) in whichthe 0.4 kb Hpa I-Nru I fragment containing the Saccharomyces cerevisiaeLEU2 gene was deleted and, in addition, contains the Saccharomycescerevisiae TPI1 promoter and the TPI1 terminator flanking an Eco RI-XbaI directional cloning site such that the a DNA insert is transcribed inthe same direction as the Schizosaccharomvces pombe POT1 gene (Norris etal., ibid.). Plasmid pMT-636 has been described in WO 89/01968 and WO90/10075, which are incorporated herein by reference in their entirety.Plasmid pMT-636 is digested with Nco I and Xba I to isolate the 9.3 kbfragment. Plasmid pMT-636 is also digested with Nco I and Eco RI toobtain the 1.6 kb fragment. The two fragments from pMT-636 are ligatedwith the Eco RI-Xba I fragment. A plasmid containing the signalsequence-Kunitz-type inhibitor domain fragment in the correctorientation is transformed into S. cerevisiae MT-663 (a/α Δtpi/Δtpipep4-3/pep4-3). Transformants were selected for growth on glucose as thesole carbon source, and cultivated in YEPD media. Transformants areassayed for activity as described in Example 4. The Kunitz-typeinhibitor is purified as described in Example 5.

B. Expression of the Kunitz-Type Inhibitor Domains of TFPI-2 ComprisingAmino Acids 34 through 152 of SEQ ID NO:2

A DNA construct encoding Kunitz-type inhibitor domains of TFPI-2comprising the amino acid sequence of SEQ ID NO:2 from glutamic acid,amino acid number 34, through lysine, amino acid number 152, isamplified from human genomic DNA as described in Example 1 usingoligonucleotide primers M-2161 and M-2162 (SEQ ID NO:5 and SEQ IDNO:11). The resulting PCR-generated fragment is gel-purified and joinedto the signal sequence as described above. The plasmid intermediatecomprising the synthetic signal sequence and TFPI-2 coding sequence inthe vector pTZ19R is used to obtain the signal sequence-TFPI-2 fragmentfor the construction of the yeast expression vector. The Eco RI-Xba Ifragment from the plasmid intermediate encoding the signalsequence-TFPI-2 is subcloned into the yeast expression vector MT-636 asdescribed above. A candidate plasmid having the correct insert istransformed into Saccharomyces cerevisiae strain MT-663 as describedabove.

Selected transformants are assayed for activity as described in Example4. The Kunitz-type inhibitor is purified as described in Example 5.

C. Expression of Kunitz-Type Inhibitor Domains of TFPI-2 ComprisingAmino Acids 34 through 211 of SEQ ID NO:2

A DNA construct encoding Kunitz-type inhibitor domains of TFPI-2comprising the amino acid sequence of SEQ ID NO:2 from glutamic acid,amino acid number 34, through alanine, amino acid number 211 isconstructed by first digesting plasmid pJ-2-11/pUC19 with Bgl II andHind III to obtain a 528 bp Bgl II-Hind III fragment encoding the threeKunitz-type domains. The Kunitz-type inhibitor domains coding sequencefrom pJ-2-11/pUC19 is joined to the synthetic signal sequence (SEQ IDNO:7) by replacing the TFPI-2 coding sequence in the plasmidintermediate described in Example 3B. The plasmid intermediate isdigested with Bgl II and Xba I to isolate the vector-containingfragment. The Bgl II-Xba I vector containing fragment is ligated withthe Bgl II-Hind III fragment from pJ-2-11/pUC19 and a Hind III-Xba Ilinker containing a translation stop codon. A plasmid containing thesynthetic signal sequence joined in the proper orientation with theTFPI-2 coding sequence is identified.

The Eco RI-Xba I fragment from the plasmid intermediate encoding thesignal sequence-TFPI-2 is subcloned into the yeast expression vectorMT-636 as described above. A candidate plasmid having the correct insertis transformed into Saccharomyces cerevisiae strain MT-663 as describedabove.

Selected transformants are assayed for activity as described in Example4. The Kunitz-type inhibitor is purified as described in Example 5.

Example 4 Activity Assays

A. Trypsin Inhibitory Activity Assay on Mammalian Cell CultureSupernatants

Conditioned media from cells expressing Kunitz-type inhibitors wasassayed for trypsin inhibitor activity. For each clone, 20-100 μl ofconditioned medium was added to a solution containing 2.4 μg/ml trypsin(Worthington Biochemical, Freehold, N.J.) in 100 mM NaCl, 50 mM Tris (pH7.4) to give a final volume of 300 μl. The reactions were incubated at23° C. for 30 minutes after which 20 μl of 10 mM chromogenic substrateS-2251 (D-Val-Leu-Lys-Nan; Chromogenix, AB, Mölndal, Sweden) was addedto a final concentration of 0.6 mM. The residual trypsin activity wasmeasured by absorbance at 405 nm.

B. Activity Assay on Yeast Culture Supernatants

Trypsin inhibitory activity is measured on the spent media from culturesof yeast transformants described in Example 3 by diluting 3.2 μl of eachspent medium sample with 80 μl of assay buffer (50 mM Tris HCl, pH 7.4,100 mM NaCl, 2 mM CaCl₂, 0.1% w/v PEG 20,000). The diluted supernatantis added to 80 ml of 133 nM bovine trypsin (Novo Nordisk A/S) diluted inassay buffer, and the mixture is incubated for 10 minutes at roomtemperature. After incubation, 100 ml of 1.8 mM peptidyl nitroanilidesubstrate S2251 (D-Val-Leu-Lys-Nan; Kabi) diluted in assay buffer isadded to each sample, and the samples are incubated with the substratefor 30 minutes. Trypsin inhibitory activity is indicated by a colorlesssolution. A control reaction, which results in a yellow solution, isproduced by a supernatant from a yeast strain not expressing anyKunitz-type inhibitor.

Example 5 Purification of Kunitz-Type Inhibitors

A. Purification of Kunitz-Type Inhibitors from Transfected MammalianCell Culture Supernatants

Recombinant TFPI-2 was purified from conditioned medium by sequentialapplication of heparin agarose, MONO Q, MONO S and SUPEROSE 12chromatography as described in more detail below. Approximately fiveliters of conditioned serum-free media was adjusted to pH 7.5 with 1 NNaOH and filtered through a 0.22-μm filter. A 2.6×35 cm heparinsepharose column (Pharmacia Biotech Inc., Piscataway, N.J.) wasequilibrated at 4° C. with Buffer A (50 mM Tris-HCl (pH 7.5), 10%glycerol). The filtered media was applied to the equilibrated column ata flow rate of 3 ml/min. Following sample application, the column waswashed with Buffer A containing 0.2 M NaCl. TFPI-2 activity, as judgedby its ability to inhibit trypsin (Example 4A), was eluted from thecolumn with Buffer A containing 1 M NaCl. The eluent from the heparinsepharose column was dialyzed at 4° C. against 25 mM Tris-HCl (pH 7.5),10% glycerol. The retentate was subjected to FPLC (Pharmacia BiotechInc.) on a 5×50 mm column containing an anion exchanger with quaternaryamine groups crosslinked to a beaded hydrophylic resin such as a MONO Q(MONO Q HR 5/5; Pharmacia Biotech Inc., Piscataway, N.J.) or the likethat had been equilibrated with 25 mM Tris-HCl (pH 7.5), 10% glycerol atroom temperature. TFPI-2 was eluted from the column in a linear NaClgradient (from 0-0.5 M NaCl) at a flow rate of 1 ml/min. The TFPI-2fractions were pooled and dialyzed against 25 mM sodium citrate (pH5.0), 10% glycerol. The retentate was then subjected to FPLC at roomtemperature on a 5×50 mm column containing a cation exchanger withcharged sulfonic groups coupled to a beaded hydrophylic resin such asMONO S (MONO S HR 5/5, Pharmacia Biotech Inc.) or the like at a flowrate of 0.5 ml/min. TFPI-2 activity was eluted from the MONO S columnwith a gradient elution from 25 mM sodium citrate (pH 5.0), 10% glycerolto 25 mM Tris-HCl (pH 7.5), 10% glycerol, 1 M NaCl. Fractions containingTFPI-2 activity were pooled and concentrated to approximately 1 ml byultrafiltration. The concentrated samples were subjected to FPLC acrossa cross-linked agarose gel filtration matrix having a porosity suitablefor the separation of proteins from 1×10³ to 3×10⁵ MW such as SUPEROSE12 (Pharmacia Biotech Inc., Piscataway, N.J.) or the like at roomtemperature in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl. Fractions elutedfrom the FPLC with TFPI-2 activity were subjected to SDS-PAGE, and purefractions were pooled and stored at −80° C.

B. Purification of Kunitz-Type Inhibitors from Yeast CultureSupernatants

Kunitz-type inhibitors are purified from yeast culture supernatantsessentially as described by Norris et al. (ibid.; which is incorporatedherein by reference). Selected transformants are grown in 10 liters ofYEPD for approximately 40 hours at 30° C. until an OD₆₀₀ ofapproximately 25 has been reached. The culture is centrifuged, and thesupernatant is decanted.

For purification, a 300 ml-1000 ml aliquot of supernatant is adjusted topH 2.3 and applied to a column holding 8 ml of S-Sepharaose(Pharmacia-LKB Biotechnology AS, Alleroed, Denmark) that has beenpreviously equilibrated with 20 mM Bicine, pH 8.7 (Sigma Chemical Co.,St. Louis, Mo.). After the column has been extensively washed with 20 mMBicine, pH 8.7, the Kunitz-type inhibitor is eluted with 30 ml of 20 mMBicine, pH 8.7 containing 1 M NaCl. The eluted material is desalted byapplication to a Sephadex G-25 column (Pharmacia-LKB Biotechnology AS,Alleroed, Denmark; 2.5×30 cm) that has been equilibrated with 20 mMNH₄HCO₃, pH 7.8. The Kunitz-type inhibitor is eluted with 20 mM NH₄HCO₃,pH 7.8.

The Kunitz-type inhibitor is further purified and concentrated bychromatography on a Mono S column (Pharmacia-LKB Biotechnology AS,Alleroed, Denmark; 0.5×5 cm) equilibrated with 20 mM Bicine, pH 8.7.After washing with the equilibration buffer at 2 ml/min for 10 minutes,gradient elution of the Kunitz-type inhibitor is carried out over twelveminutes at 1 ml/min from 0-0.6 M NaCl in the equilibration buffer. Peaksamples are pooled, and the Kunitz-type inhibitor is purified usingreverse phase HPLC on a Vydac 214TP510 column (Mikro-lab, Aarhus,Denmark; 1.0×25 cm) with a gradient elution at 4 ml/min from 5% A (0.1%trifluoroacetic acid (TFA) in water) to 45% B (0.7% TFA in acetonitrile)in 20 minutes. The purified product in lyophilized in water, andinhibitor activity is measured.

Kunitz inhibitor activity is measured using the method essentiallydescribed by Norris et al. (ibid.). Briefly, various fixedconcentrations of the Kunitz-type inhibitor are incubated in thepresence of 0.24 μg/ml of porcine trypsin (Novo Nordisk A/S, Bagsvaerd,Denmark), 12.8 CU/l human plasmin (Kabi, Stockholm, Sweden) or 0.16nkat/ml human plasma kallikrein (Kabi) in 100 mM NaCl, 50 mM Tris HCl,pH 7.4. After a 30 minute incubation the residual enzymatic activity ismeasured by the cleavage of a substrate solution containing 0.6 mM ofeither of the chromogenic peptidyl nitroanilide trypsin/plasminsubstrates S2251 (D-Val-Leu-Lys-Nan; Kabi) or S2302 (D-Pro-Phe-Arg-Nan;Kabi) in assay buffer. The samples are incubated for 30 minutes afterwhich the absorbance of each sample is measured at 405 nm. Plasmin ortrypsin activity is measured as a decrease in absorbance at 405 nm. Fromthe results, the apparent inhibition constant Ki is calculated.

Example 6 Effect of Recombinant TFPI-2 on the Amydolytic Activities ofHuman Thrombin, Human Factor XA and a Complex of Human FactorVIIA/Tissue Factor

A. Thrombin Amidolytic Activity Assay

The ability of recombinant TFPI-2 to inhibit the amidolytic activity ofhuman thrombin was determined by a colometric assay using human thrombin(prepared as described by Pedersen, et al., J. Biol. Chem. 265:16786-16793, 1990; which is incorporated by reference herein in itsentirety) and various concentrations of recombinant TFPI-2. The assaywas set up in a microtiter plate format. Reactions of 200 μl wereprepared in the wells of the microtiter plate. The reaction mixturescontained various concentrations of recombinant TFPI-2 and 20 nM humanthrombin in 50 mM Tris-HCl (pH 7.5), 0.1% BSA, 5 mM CaCl₂. The reactionswere incubated at 37° C. for 15 minutes. Following incubation, 50 μl of10 mM the chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-p-nitroanilide,Chromogenix, AB, Mölndal, Sweden) was added to each well. The absorbanceat 405 nm was determined in a kinetic microplate reader (Model UVMAX,Molecular Devices). Recombinant TFPI-2 was shown to have no effect onthe amidolytic activity of human thrombin towards S-2238

B. Human Factor Xa Amidolytic Assay

The ability of recombinant TFPI-2 to inhibit the amidolytic activity offactor Xa was determined by a colorimetric assay as described aboveusing 20 nM human factor Xa (prepared as described by Kondo, and Kisiel,Blood 70, 1947-1954, 1987; which is incorporated by reference herein inits entirety) in place of the 20 nM human thrombin described above. Thereactions were set up and incubated as described above replacing thehuman thrombin with human factor Xa. Following incubation, 50 ml of 10mM of the chromogenic substrate S-2222(Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, Chromogenix, AB, Mölndal,Sweden) was added to each well. The absorbance at 405 nm was determinedin a kinetic microplate reader (Model UVMAX, Molecular Devices).Recombinant TFPI-2 was shown to weakly inhibit the amidolytic activityof 20 nM factor Xa towards the chromogenic substrate S-2222 in adose-dependent manner.

C. Human Factor VIIa/Tissue Factor Amidolytic Assay

The ability of recombinant TFPI-2 to inhibit the amidolytic activity offactor VIIa/tissue factor complex was determined by a colorimetric assayusing 70 nM recombinant, truncated, human tissue factor apoproteinconsisting of the 219-amino acid extracellular domain (TF₁₋₂₁₉)(prepared as described by Paborsky, et al., J. Biol. Chem. 266:21911-21916, 1991; which is incorporated herein in its entirety)provided by Gordon Vehar (Genentech Inc., South San Francisco, Calif.),and 20 nM recombinant human factor VIIa (prepared as described byPedersen, et al., Biochemistry 28: 9331-9336, 1989; which isincorporated by reference herein in its entirety) provided by PeterWildgoose (Novo Nordisk A/S, Bagsvaerd, Denmark) in place of the 20 nMhuman thrombin described above. The assay was set up and incubated asdescribed above replacing the human thrombin with human factor VIIa andTF-₁₋₂₁₉. Following incubation, 50 ml of 10 mM chromogenic substrateS-2288 (H-D-Ile-Pro-Arg-p-nitroanilide, Chromogenix, AB) was added toeach well. The absorbance at 405 nm was determined in a kineticmicroplate reader (Model UVMAX, Molecular Devices). Recombinant TFPI-2was shown to inhibit the amidolytic activity of 20 nM factor VIIa-tissuefactor towards the chromogenic substrate S-2288 in a dose-dependentmanner.

Example 7 Amino Acid Sequence Analysis

Automated amino acid sequencing was performed in a gas vapor sequenator(Beckman Instruments; Model LF 3000 or the like) equipped with anon-line phenylthiohydantoin analyzer. The phenylthiohydantoin peaks wereintegrated using SYSTEM GOLD software provided with the sequenator.Approximately 100 picomoles of protein were subjected to sequenceanalysis. Amino-terminal amino acid sequence analysis of a singlepreparation of recombinant TFPI-2 indicated a major sequence (˜70%) ofAsp-Ala-Ala-Gln-Glu-Pro-Thr-Gly-Asn-Asn (SEQ ID NO:12) and a minorsequence (˜30%) of Ala-Gln-Glu-Pro-Thr-Gly-Asn-Asn (SEQ ID NO:13),suggesting either alternative cleavage sites by the signal peptidase, orpossible amino-terminal degradation by exopeptidases during itspurification.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated human Kunitz-type inhibitor that inhibits plasmin in amammal and wherein DNA sequence encoding the human Kunitz-type inhibitorhybridizes to nucleotides 138-305 of SEQ ID NO:1 under highly stringenthybridization conditions.
 2. The isolated human Kunitz-type inhibitor ofclaim 1 wherein any differences between the human Kunitz-type inhibitorand amino acid number 34 to amino acid number 89 of SEQ ID NO:2 are dueto conservative amino acid substitutions.
 3. A pharmaceuticalcomposition comprising the human Kunitz-type inhibitor of claim
 1. 4.The pharmaceutical composition of claim 3 wherein the human Kunitz-typeinhibitor is isolated from E. coli.
 5. A DNA construct comprising afirst DNA segment, wherein the first DNA segment is the DNA sequence ofclaim 1, operably linked to additional DNA segments required for theexpression of the first DNA segment.
 6. A host cell comprising the DNAconstruct of claim 5 wherein the host cell expresses the humanKunitz-type inhibitor encoded by the first DNA segment.
 7. The host cellof claim 6 wherein the host cell is E. coli.
 8. A method for producinghuman Kunitz-type inhibitor comprising: culturing a cell according toclaim 6; and isolating the human Kunitz-type inhibitor produced by thecell.
 9. The method of claim 8 wherein the cell is E. coli.
 10. Anisolated DNA sequence that hybridizes to nucleotides 138-305 SEQ ID NO:1under highly stringent hybridization conditions, wherein the isolatedDNA sequence encodes a human Kunitz-type inhibitor that inhibits plasminin a mammal.
 11. The isolated DNA sequence of claim 10 wherein anydifferences between the encoded human Kunitz-type inhibitor and aminoacid number 34 to amino acid number 89 of SEQ ID NO:2 are due toconservative amino acid substitutions.
 12. An isolated human Kunitz-typeinhibitor that inhibits plasmin in a mammal and wherein DNA sequenceencoding the human Kunitz-type inhibitor hybridizes to nucleotides39-743 of SEQ ID NO:1 under highly stringent hybridization conditions.13. The isolated human Kunitz-type inhibitor of claim 12 wherein anydifferences between the human Kunitz-type inhibitor and amino acidnumber 1 to amino acid number 235 of SEQ ID NO:2 are due to conservativeamino acid substitutions.
 14. A pharmaceutical composition comprisingthe human Kunitz-type inhibitor of claim
 12. 15. The pharmaceuticalcomposition of claim 14 wherein the human Kunitz-type inhibitor isisolated from E. coli.
 16. A DNA construct comprising a first DNAsegment, wherein the first DNA segment is the DNA sequence of claim 12,operably linked to additional DNA segments required for the expressionof the first DNA segment.
 17. A host cell comprising the DNA constructof claim 16 wherein the host cell expresses the human Kunitz-typeinhibitor encoded by the first DNA segment.
 18. The host cell of claim17 wherein the host cell is E. coli.
 19. A method for producing humanKunitz-type inhibitor comprising: culturing a cell according to claim17; and isolating the human Kunitz-type inhibitor produced by the cell.20. The method of claim 19 wherein the cell is E. coli.
 21. An isolatedDNA sequence that hybridizes to nucleotides 39-743 SEQ ID NO: 1 underhighly stringent hybridization conditions, wherein the isolated DNAsequence encodes a human Kunitz-type inhibitor that inhibits plasmin ina mammal.
 22. The isolated DNA sequence of claim 21 wherein anydifferences between the encoded human Kunitz-type inhibitor and aminoacid number 1 to amino acid number 235 of SEQ ID NO:2 are due toconservative amino acid substitutions.